Microsporogenesis of Rps8/rps8 heterozygous soybean lines

Maria Andrea Ortega • Anne E. Dorrance

Received: 14 November 2010 / Accepted: 25 March 2011 / Published online: 7 April 2011

� The Author(s) 2011. This article is published with open access at Springerlink.com

Abstract Phytophthora root and stem rot caused by

Phytophthora sojae, is one of the most damaging

diseases of soybean, for which management is

principally done by planting resistant cultivars with

race specific resistance which are conferred by Rps

(Resistance to Phytophthora sojae) genes. The Rps8

locus, identified in the South Korean landrace PI

399073, is located in a 2.23 Mbp region on soybean

chromosome 13. In eight cv. Williams (rps8/

rps8) 9 PI 399073 (Rps8/Rps8) populations, this

region exhibited strong segregation distortion. In a

cross between the South Korean lines PI 399073

(Rps8/Rps8) and PI 408211B (multiple Rps genes)

this region segregated in a Mendelian fashion. In this

study, microsporogenesis was evaluated to identify

meiotic abnormalities that may be associated with

the segregation distortion of the Rps8 region. Pollen

was collected from greenhouse-grown plants of the

parental genotypes: Williams, PI 399073, and PI

408211B; as well as selected Rps8/rps8 RILs

from Williams 9 PI 399073 BC4F2:3 and PI 399073 9

PI 408211B F4:5 populations. There were no differ-

ences for pollen viability among the genotypes.

However, for PI 399073, a mix of dyads, triads,

tetrads and pentads was observed. A high frequency

of meiotic abnormalities including fragments,

laggards, multinucleated microspores; and micro-

cytes containing DNA was also observed in Rps8/

rps8 Williams 9 PI 399073 BC4F2:3 RILs. These

meiotic abnormalities may contribute to the high

degree of segregation distortion present in the

Williams 9 PI 399073 populations.

Keywords Microsporogenesis � Segregation

distortion � Soybean � Pollen � Resistance gene

Introduction

Glycine is a genus of leguminous plants, which

includes the cultivated soybean (Glycine max [L.]

Merr.), the wild annual soybean (Glycine soja Sieb.

& Zucc.), as well as a number of perennial species

(Kollipara et al. 1995; Hymowitz 2004). Phytoph-

thora sojae Kaufm. & Gerd. is an oomycete pathogen

of soybeans, causing root and stem rot in older plants,

and damping-off of seedlings. Annual worldwide

losses to Phytophthora root and stem rot can reach

US $1–2 billion (Wrather et al. 2001; Wrather and

Koenning 2006). The disease is managed through the

deployment of single genes (Rps genes) that confer

resistance to P. sojae. Currently fourteen Rps alleles

have been reported at eight different loci. The Rps8

locus was identified in the South Korean landrace PI

399073 (Dorrance and Schmitthenner 2000), and was

assigned to the soybean molecular linkage group

M. A. Ortega � A. E. Dorrance (&)

Department of Plant Pathology, The Ohio State

University, Wooster, OH 44691, USA

e-mail: dorrance.1@osu.edu

123

Euphytica (2011) 181:77–88

DOI 10.1007/s10681-011-0422-1

(MLG) F (Gordon et al. 2006) corresponding to

chromosome 13 of G. max.

Multiple mapping populations were developed in

recent years by crossing PI 399073 and the cultivar

Williams, which is considered as the universal

susceptible genotype to P. sojae. These segregating

populations were advanced for several generations

for the purpose of the identification of molecular

markers for the development of breeding material

carrying the Rps8 locus, and to assist in the cloning of

the gene. Each population was phenotyped for

resistance to P. sojae and genotyped with a set of

markers on chromosome 13. The resistance pheno-

type was associated with markers in the Rps8 locus

region (Ortega et al. 2010). However, the expected

phenotypic and genotypic segregation ratios were

always highly skewed (Ortega et al. 2008, 2010). In

each of the populations derived from Williams 9 PI

399073 crosses, an excess of Rps8/Rps8 homozy-

gous RILs and Rps8/rps8 heterozygous RILs were

obtained at the expense of the rps8/rps8 homozygous

RILs.

Segregation distortion is not uncommon in map-

ping populations, and is one of the several factors that

influence the precision of genetic mapping. It has

been shown that both the genetic distance between

markers and the order of the markers on linkage

groups could be affected by this phenomenon

(Lorieux et al. 1995a, b). In soybean, 18 regions on

ten different linkage groups where high levels of

segregation distortion occur were previously

described (Yamanaka et al. 2001), including the

soybean root fluorescence locus Fr1 on chromosome

9 (MLG K) (Jin et al. 1999). The mechanisms

involved in segregation distortion are not well

understood. However gametophytic factors, compe-

tition among gametes, or the abortion of gametes

have all been proposed (Lu et al. 2000, 2002; Lyttle

1991; Matsushita et al. 2003).

Continued development of virulent pathotypes of

P. sojae that can infect plants with Rps resistance

genes (Grau et al. 2004) and the limited number of

effective Rps genes that are currently deployed in US

cultivars, makes the quest for novel sources of

resistance a high priority. Sources of resistance to

host-specific plant pathogens are usually found in the

regions of greatest differentiation of host species

(Leppik 1970). In the case of Phytophthora root and

stem rot, sources of resistance have been identified

in both Chinese and South Korean germplasm

(Dorrance and Schmitthenner 2000; Kyle et al. 1998;

Lohnes et al. 1996). However, phenomena like

segregation distortion can complicate the develop-

ment of resistant cultivars when genes are introgres-

sed from novel sources of resistance. In the case of

Rps8, segregation distortion in populations derived

from PI 399073 represents a major challenge to fine

map and clone this gene. Identifying the mechanisms

that contribute to the segregation distortion of Rps8 is

crucial for the identification of breeding strategies

that will expedite the use of this gene for the

management of Phytophthora root and stem rot of

soybeans.

In plant development, microsporogenesis is the

cellular division that produces haploid microspores

which develop into pollen grains, and this is also an

important process where meiotic abnormalities may

be detected. A large number of microspores are

produced in each anther, making them a feasible

target for the evaluation of the different meiotic

phases. Normal microsporogenesis in soybean has

been described (Albertsen and Palmer 1979), and

serves as a useful guide for the evaluation of

abnormalities. Structural changes in chromosomes

that effect the production of normal gametes, such as

inversions and translocations (Mahama et al. 1999;

Palmer et al. 2000) have also been previously

described in soybean.

To produce pollen grains, male gametogenesis

starts with the division of a diploid sporophyte that

gives rise to both the tapetum and pollen mother cells

(PMCs) (McCormick 1993). The later cells undergo

meiosis and give rise to tetrad cells that are released

as microspores when the callose is degraded by the

enzyme callase produced by the tapetum. The

microspores undergo mitosis to generate pollen

grains containing the larger vegetative cell and the

small generative cell. The irregularities reported in

soybean male meiosis include: chromosome associ-

ations, abnormal spindles, precocious chromosome

migration, chromosome stickiness, chromosome frag-

ments, laggards, bridges, micronuclei, cytokinesis

failure, and production of microcytes (Bione et al.

2000, 2002, 2005; Kumar and Rai 2006; Palmer et al.

2000). In general, abnormal male meiosis tends to

have an outcome of partial or total pollen sterility.

This is especially important in soybean, as this genus

self fertilizes and genotypic factors associated with

78 Euphytica (2011) 181:77–88

123

those pollen grains would not be passed to the next

generation.

Due to the high level of segregation distortion

across the numerous crosses of PI399073 and G. max

cultivars, a comparative study of Rps8/rps8 lines

from two parental combinations, Williams 9 PI

399073 and PI 399073 9 PI 408211B was initiated.

Our objective was to determine if male gametogen-

esis abnormalities occur in Rps8/rps8 lines which

originated from populations with a high degree of

segregation distortion as well as examine populations

where this locus segregates in a true Mendelian

fashion. For each genotype, the targets were meiosis

I and II, microspores, and mature pollen grains.

Materials and methods

Plant material

A BC4F2:3 population consisting of 30 lines was

generated by backcrossing the region containing the

Rps8 locus from PI 399073 into cultivar Williams

(recurrent parent). BC4 seeds were advanced by

single-seed-descent to generate F2:3 seeds. In a

previous study, the population was phenotyped for

resistance to P. sojae through hypocotyl inoculation.

P. sojae isolate OH25, virulent to plants carrying the

Rps genes 1a, 1b, 1c, 1k, and 7, was used to identify

lines carrying the resistance locus Rps8 from PI

399073 in the BC4F2:3. In addition, a F4:5 population

consisting of 152 recombinant inbred lines (RILs)

was generated by crossing PI 399073 and PI

408211B, F2 seeds were advanced by single-seed-

descent. The phenotypic data for disease resistance

was obtained by inoculation with P. sojae isolate

BUTMU. This isolate has a compatible interaction

(susceptible response) with the Rps genes in PI

408211B, and an incompatible interaction (resistance

response) in PI 399073. Lines with a heterozygous

phenotype were genotyped with 72 SSR and SNP

markers in the Rps8 region, and four lines were

selected from each population for this study, these

lines were heterozygous for the resistance phenotype

and molecular markers located between the SSRs

Satt114 and Satt362 (Table 1).

Seeds from each heterozygous line and parental

genotype were planted in 2-l pots of sterilized soil

mixture. Based on earlier genotypic data, recombi-

nant inbred lines (RILs) 1403, 1404, 1412, and 1413

from the BC4F2:3; and RILs 37, 50, 58, and 143

from the F4:5 populations were selected for this

Table 1 Genotype of the Rps8 region on the selected BC4F2:3 lines and F4:5 RILs

Chromosome 13 Williams 9 PI 399073 BC4F2:3 PI 399073 9 PI 408211B F4:5

Rps8/rps8 lines Rps8/rps8 lines

1403 1404 1412 1413 37 50 58 143

Position Mbpa Marker 1 2 1 2 1 2 3 4 1 1 2 1 2 1 1 2 3

27.71 Satt114 A A A A A A A A A C C H H H H H H

28.21 F420_18 H H H H H H H H H H H H H H H H H

28.41 Satt334 H H H H H H H H H H H H H H H H H

28.84 F336_18 H H H H H H H H H H H H H H H H H

29.01 98FA16.2 H H H H H H H H H H H H H H H H H

29.03 F336_01 H H H H H H H H H H H H H H H H H

29.27 F396_08 H H H H H H H H H H H H H H H H H

29.30 AC15916.2 H H H H H H H H H H H H C H H H H

32.86 Satt362 A A A A A A A A A H H H H H H H H

These plants are the progeny of Rps8/rps8 plants, and the maintenance of heterozygosity was confirmed on genomic DNA isolated

from the seedlings. Williams homozygous locus (A), Williams/PI 399073 heterozygous locus (H), PI 408211B homozygous locus

(C), and PI 408211B/PI 399073 heterozygous locusa Position based on Williams82 8X assembly (Mbp) (available at http://www.phytozome.net/soybean.php)

Euphytica (2011) 181:77–88 79

123

experiment (Ortega et al. 2010). Plants were grown at

27�C with 14 h daylight period (with supplemental

lighting provided); watered twice every day, and

supplemented with 100 ppm 20N:20P:20K green-

house fertilizer. Studies were from January to March

2009.

DNA isolation and genotyping

Genomic DNA was extracted using a modification

of the protocol described by Keim et al. (1988).

Cotyledons from 3 week-old seedlings were collected

in 10 9 10 cm2 reclosable plastic bags (Uline, Inc.,

Philadelphia, PA) and stored at 4�C until processing.

Tissue was ground in CTAB buffer: 100 mM Tris–

HCl pH 8.0, 1.4 mM NaCl, 2.0% CTAB (hexadecyl-

trimethyl-ammonium bromide), and 20 mM EDTA

pH 8.0. One milliliter of the extraction buffer was

added to the plastic bag, and the tissue was macerated

using a hand-held roller (BIO-RAD, Hercules, CA).

The suspension was transferred to a 2.0 ml tube and

incubated at 65�C for 1 h, and mixed vigorously

every 15 min. The sample was cooled to 27�C, and

an equal volume of 24:1 (v/v) chloroform-isoamyl

alcohol (Sigma Chemical Co., St. Louis, MO)

was added. An emulsion formed after inverting the

tubes several times, followed by centrifugation at

10,000 rpm for 10 min in a table-top microcentri-

fuge. The supernatant was transferred to a 2.0 ml

tube, and the DNA was precipitated from the solution

by adding 99% isopropyl alcohol and centrifuged at

10,000 rpm for 10 min. The supernatant was dis-

carded and the DNA pellet was washed with 70%

ethyl alcohol. The DNA pellet was air dried overnight

and resuspended in 500 ll of TE buffer (10 mM,

1 mM EDTA pH 8.0). RNA was removed by

treatment with 2.0 ll of 5 mg/ml Ribonuclease A

(Sigma–Aldrich, St. Louis, MO) were added to the

reactions and incubated at 37�C for 1 h. DNA was

quantified by spectrophotometry using NanoDrop

(Thermo Fisher Scientific, Waltham, Massachusetts,

USA) following the manufacturer instructions, and

the samples were stored at -20�C.

To verify that each RIL carried heterozygous

genotypes, each line was screened with eight SSR

markers across the Rps8 region. The markers Satt114,

Satt334, Satt362 located on chromosome 13 (MLG F)

on the soybean consensus map (Cregan et al. 1999);

AC15916, 98FA16; and F420_18, F336-01, and

F336_18 developed from sequences from Williams82

BAC clones and mapped to chromosome 13 were

used for genotyping. Template DNA was diluted to

50 ng/ll in TE buffer and stored at -20�C. The PCR

amplification was done in a 12.5 ll reaction mixture

containing 19 Green Go Taq Flexi Buffer (Promega,

Madison, WI), 2 mM MgCl2 (Promega), 200 mM of

each deoxynucletide (Promega), 200 nM of each

primer, 1 U Go Taq DNA polymerase (Promega),

and 50 ng of genomic DNA. All PCR reactions were

carried out on a DNA Engine Tetrad 2 Peltier

Thermal Cycler (BioRad, Hercules, CA). The thermal

conditions were 94�C for 5 min; ten cycles of touch-

down PCR: 94�C for 45 s, 60–50�C (decreasing 1�C

per cycle) for 45 s, and 72�C for 1 min; followed by

24 cycles with annealing temperature of 50�C; and

final extension at 72�C for 10 min. PCR products

were analyzed on 4.0% agarose 3:1 HRB (Amresco,

Solon, OH). Agarose was dissolved in 19 RapidRun

agarose buffer (USB, Cleveland, Ohio), pre-stained

with 0.5 lg/ml of ethidium bromide (Sigma–Aldrich,

St. Louis, MO) and cast in 20 9 25 cm trays (Fisher

Scientific, Pittsburgh, PA). Ten microliter amplicons

were electrophoresed in 19 RapidRun agarose buffer

for 25 min at 250 V. Electrophoresed gels were

visualized and digitally photographed.

Pollen viability and germination

Open flowers were collected between 9:00 and 11:00

a.m. during the first 3 weeks of flowering. Three

flowers were collected from each plant, three times a

week. Each set of anthers was dissected and dusted

onto pollen germination medium (Gwata et al. 2003)

and incubated at 27�C for 18 h. A minimum of 100

pollen grains per anther were observed for germina-

tion under a S6D Stereozoom microscope (Leica

Microsystems Inc., Deerfield, Illinois, USA). A grain

was classified as germinated if a recognizable pollen

tube, at least 20 lm long was present. Pollen

viability was assessed with Lugol’s solution (Elec-

tron Microscopy Sciences, Hatfield, PA), consisting

of 5% iodine and 10% potassium iodide, and this

staining detects starch content. The same set of

anthers used to determine percent germination were

placed in 100 ll of Lugol’s solution on a

25.4 9 76.2 mm slide (Becton–Dickinson Labware,

Franklin Lakes, NJ). The slide was covered with a

22 9 40 mm cover glass (Daigger, Vernon Hills, IL)

80 Euphytica (2011) 181:77–88

123

and visualized under a binocular DME light micro-

scope with the 209 magnification objective (Leica

Microsystems Inc., Deerfield, IL). Pollen grains

which were stained dark brown to black were

considered viable.

Cytological analysis of male meiosis

In this study acetic carmine staining was used for

visualization of the chromosomes (Schreiber 1954).

Immature flower bud clusters were collected

between 9:00 and 12:00 a.m., the stage reported

for meiotic analysis in soybeans (Bione et al. 2003;

Mahama et al. 1999; Palmer et al. 2000). Flower

buds from single plants were placed in 2.0 ml

microcentrifuge tubes containing 1.5 ml of formalin-

aceto-alcohol mixture (Ricca Chemical Company,

Arlington, TX) for fixation. The samples were

incubated at 27�C for 24 h and stored at 4�C until

assayed.

Anthers were dissected and transferred to 0.2 ml

tubes containing 0.75% acetic carmine (Carolina

Biological Supply Company, Burlington, NC). Car-

mine was dissolved in 45% acetic acid, and it served

the double purpose of fixation and staining; acetic

acid penetrates membranes rapidly, and carmine is

insoluble in chromatin. The staining was enhanced by

adding 2 ll of 10% w/v ferric chloride solution

(Sigma Chemical Co., St. Louis, MO). Dissected

anthers were incubated at 70�C for 8 h and main-

tained at 27�C for another 24 h. The anthers were

blotted on Kimwipes (Kimberly-Clarke, Roswell,

GA) and placed on a 25.4 9 76.2 mm slide (Bec-

ton–Dickinson Labware, Franklin Lakes, NJ) con-

taining 100 ll of mounting media (Rattenbury 1956).

The slide was covered with a 22 9 40 mm cover

glass (Daigger, Vernon Hills, IL). The slides were

placed on the dissecting scope and each anther was

crushed, by applying pressure on the cover glass with

a dissecting needle, until the anther wall broke and

meiotic cells were released. The preparations were

sealed with nail polish.

The preparations were viewed under a binocular

DME light microscope (Leica Microsystems Inc.,

Deerfield, Illinois, USA) at 10009 magnification,

and photographed using a Nikon digital sight

DS-SM camera and DS-L1 computer (Nikon Corp.,

Japan).

Results

Pollen viability

One plant from each line and parents was used for

evaluation of pollen viability, this was done so that it

would be possible detect variation for this parameter

between the different collection times, and at the

same time leave enough immature flowers for the

cytogenetic studies. A mean of 90% of the pollen

grains stained dark brown with Lugol’s solution

indicating viability (data not shown). In addition,

there was no significant difference among plants for

pollen germination on the same sampling day, but a

significant (P = 0.05) difference was found between

sampling days for the same plant (Fig. 1). Pollen

collected from flowers produced on the first week of

the reproductive stage, independently of the plant

evaluated, had germination percentages lower than

55%. The percentage of pollen grains that germinated

increased in the second week, and was maintained

above 85% during the third week of flowering.

Cytogenetics

During the microsporogenesis process in PI 399073

and RILs from the BC4F2:3 Williams 9 PI399073

several meiotic abnormalities were observed from

the stained anthers collected from immature flower

clusters. Pollen abnormalities were not found in PI

408211B nor in Williams. For the Williams 9 PI

399073 BC4F2:3 derived plants, there were 255

abnormal meiotic cells from the 371 meiotic cells

evaluated; this number was higher than the observed

in any other genotype. For the PI 399073 9 PI

408211B F4:5 lines, only 9 of 396 cells exhibited

abnormalities (Table 2). During meiosis I, the fol-

lowing abnormalities were observed: extra nucleolus

(Fig. 2a) in the pollen mother cells at prophase I,

chromosome fragments that were not part of the

metaphase plate (Fig. 2b–d), laggards present

between the two chromosomes sets at anaphase I,

and micronuclei formed between the two nuclei at

telophase I (Fig. 2f). In the fixed flower buds of the

genotypes, anthers at meiosis I were identified more

frequently than anthers at meiosis II. Similar meiotic

abnormalities were also observed in meiosis II.

The types of abnormalities of the male gametes

were determined by observation of the microspores

Euphytica (2011) 181:77–88 81

123

formed after meiosis II (Table 2). Flowers in the

same cluster were each in a different stage of

microsporogenesis, thus the characteristics of

microspores and pollen grains were noted for each

genotype (Table 3). In PI 399073, a mix of dyads,

triads, and pentads were found (Fig. 3). In this

parental genotype, tetrads comprised only 67% of

the meiotic products; and 63% of them had micro-

nuclei in at least one of the microspores. For

Williams and PI 408211B, only tetrads were

observed. In selected lines from the Williams 9 PI

399073 BC4F2:3, microspores containing micronuclei

were common (Fig. 2j, k), and ‘triads’ containing two

microspores and a microcyte were also observed

(Fig. 2i). This type of triad was only observed in the

anthers from the Rps8/rps8 Williams 9 PI 399073

BC4F2:3 RILs. Uninoculated microspores with thick

cell walls were formed in the non dehiscent anthers

from PI 399073, PI 408211B, the Rps8/rps8 Williams

9 PI 399073 BC4F2:3 RILs, and the Rps8/rps8 PI

399073 9 PI 408211B F4:5 RILs (Fig. 4a, b, e, f). In

contrast, in Williams, the majority of the microspores

were in the binucleate stage, after mitosis I (Fig. 4c);

germinated pollen grains were also common inside

the non dehiscent anthers of cultivar Williams

(Fig. 4d). A few non-viable pollen grains, not stained

with acetic carmine, were observed in the immature

anthers of all the genotypes evaluated (Fig. 4b).

Pollen grains from the anthers of Rps8/rps8 BC4F2:3

RILs were one-third the size of an average pollen

grain and contained one or more micronuclei, the

cytoplasm in these grains was not darkly stained but a

cell wall like structure was observed (Fig. 4e). These

small grains corresponded to 87% of the sterile pollen

found in the Rps8/rps8 BC4F2:3 RILs, and may be the

product of the microcytes formed in earlier stages.

However, these grains were not detected when

dehiscent anthers were used for evaluation of pollen

viability and germination, indicating that these small

grains may collapse and degrade before anthesis.

Fig. 1 Pollen germination percentage for one plant from each

line which was evaluated from the beginning of the reproduc-

tive phase (R1). Three open flowers were collected 3 days per

week for 3 week. The percentage of germinated grains was

determined after 18-h incubation in the medium described by

Gwata et al. (2003)

b

82 Euphytica (2011) 181:77–88

123

The meiotic abnormalities and meiotic products

observed for the BC4F2:3 Rps8/rps8 heterozygous

lines were also identified on the anthers of BC4F2:3

Rps8/rps8 heterozygous plants that were grown in a

preliminary study, during the summer of 2008 (June–

August). The plants evaluated on the preliminary

study included four lines from the same BC4F2:3

population evaluated on this study, including RIL

1412, and six lines from another BC4F2:3.

Discussion

In this study the male gametogenesis in Rps8/rps8

heterozygous lines and their parents was evaluated.

When pollen from the same anthers was studied in

selected heterozygous RILs and parental genotypes in

the first week of flowering, most of the grains stained

with Lugol’s solution, indicated that they were

viable. However, a low percentage of pollen germi-

nated under the test conditions across all genotypes.

The cause of poor pollen germination during the first

week of flowering in this study is unknown. Previ-

ously in soybeans, temperature, UV radiation, and

CO2 levels have been shown to affect pollen

morphology and germination (Koti et al. 2005).

Cytogenetic staining techniques were effective for

the visualization of meiotic chromosomes and detec-

tion of abnormalities during their separation during

haploidization. The products of male gametogenesis

Table 2 Pollen meiotic abnormalities in PI 399073, PI 408211B, Williams, and selected Rps8/rps8 heterozygous RILs from:

Williams 9 PI 399073 BC4F2:3, and PI 399073 9 PI 408211B F4:5 crosses

Phase Abnormalities Number of cells analyzed and cells exhibiting abnormalities

Parental genotypes Williams 9 PI 399073 BC4F2:3

Rps8/rps8 lines

PI 399073 9 PI 408211B F4:5

Rps8/rps8 lines

PI

399073

PI

408211B

Williams 1403 1404 1412 1413 37 50 58 143

No.a Ab No. A No. A No. A No. A No. A No. A No. A No. A No. A No. A

Meiosis I

Prophase I Extra-

nucleolic33 2 110 0 28 0 12 0 8 1 20 1 44 0 21 0 7 1 8 0 3 0

Metaphase

I

Fragmentsd 29 2 12 0 69 0 18 14 10 10 22 18 6 5 8 0 42 1 28 1 6 0

Anaphase I Laggardse 36 3 7 0 14 0 36 34 17 17 23 22 8 8 31 0 19 2 24 0 34 1

Telophase

I

Micronucleif Ng – 15 0 23 0 30 29 19 17 20 18 9 9 35 0 28 1 13 1 30 1

Meiosis II

Metaphase

II

Fragments 6 0 N 0 13 0 8 6 5 4 6 5 6 6 6 0 5 0 9 0 5 0

Anaphase

II

Laggards N – 3 0 5 0 N 0 2 1 4 4 4 2 N 0 3 0 N – 2 0

Telophase

II

Micronuclei 16 9 11 0 14 0 12 7 13 10 9 7 N – 9 0 6 0 10 0 4 0

Anthers from the flower clusters were collected during the first 3 weeks of flowering and were stained with acetic carminea Total pollen mother cells evaluated (No.)b Abnormal pollen mother cells (A)c Darkly stained, nuclei buds on late prophased Precocious chromosome migration to the polese Chromosome lagging between the anaphase spindlesf Condensation of the lagging chromosomesg N indicates that cells in this stage were not observed for this genotype

Euphytica (2011) 181:77–88 83

123

were also subjected to analysis, and two methods

were employed to determine the percentage of viable

pollen in each genotype. The frequency of meiotic

abnormalities was higher in Rps8/rps8 heterozygous

lines from a Williams 9 PI 399073 BC4F2:3 popula-

tion with a high degree of segregation distortion in

the Rps8 region, than in Rps8/rps8 heterozygous lines

from a PI 399073 9 PI 408211B F4:5 population in

which the Rps8 region segregated normally in a

Mendelian fashion.

Chromosome elimination affects the correct sep-

aration of chromosomes during cellular division and

has been attributed to: chromosome fragmentation,

micronucleous formation and chromatin degradation

(Subrahmanyam and Kasha 1973; Thomas 1988);

lagging chromosomes (laggards), bridges, chromo-

somes non-congregated at the metaphase plate, and

failure of chromosome migration to the poles during

anaphase (Bennett et al. 1976). Chromosome elimi-

nation has also been reported during microsporogen-

esis (Adamowski et al. 1998). In our study, the

meiotic abnormalities observed during microsporo-

genesis, accompanied by the presence of microspores

and microcytes containing micronuclei, indicates that

chromatin elimination may be a potential mechanism

influencing segregation distortion in these lines. This

Fig. 2 Meiotic

irregularities observed in

Williams 9 PI 399073

BC4F2:3 Lines. a Prophase

I, micronucleus. b,

c Metaphase I, chromosome

fragments. d Metaphase I,

chromosome not aligned at

the metaphase plate. e Late

anaphase I, laggards.

f Telophase I,

micronucleous.

g Metaphase II,

Chromosome fragments. h,

i Coenocytic tetrad,

binucleate cells. j Tetrad

cells, micronuclei.

k multinucleate microspore.

l Dyad, microcyte

84 Euphytica (2011) 181:77–88

123

mechanism does not seem to have noticeable effects

on pollen viability, because there was no correlation

between meiotic abnormalities and the percentage of

stained pollen or germinated pollen at later flowering

dates. However there was a correlation between the

frequency of meiotic abnormalities and the presence

of microcytes. In particular, RILs from the Wil-

liams 9 PI 399073 BC4F2:3 where a high level of

abnormalities were detected, the fertility of the

mature pollen was not affected, indicating that the

loss or gain of the micronuclei may not have a serious

effect on the pollen grain viability.

Many mechanisms of chromosome elimination

have been described (Singh 1993), however the

process involved in the elimination of micronuclei

as microcytes is still obscure. The elimination of

micronuclei from microspores in oat (Avena sativa

L.) was reported by Baptista-Giacomelli et al. (2000).

A micronucleus reaches the microspore wall and

separate from it by forming a bud, then the formed

microcyte give rise to a sterile pollen grain; this

process has not been described in any other species to

date. Partial genome elimination through micronuclei

and the production of aneuploid gametes was

described in plants from a natural population of

G. max (Kumar and Rai 2006). In this study tetrads

containing quiescent micronuclei were also present,

and pollen viability was not affected. The process that

Table 3 Characteristics of the meiotic products and pollen grains from PI 399073, PI 408211B, Williams, and selected Rps8/rps8

heterozygous RILs from: Williams 9 PI 399073 BC4F2:3, and PI 399073 9 PI 408211B F4:5 crosses

Male gametogenesis products Parental Genotypes Williams 9 PI 399073 BC4F2:3 PI 399073 9 PI 408211B F4:5

Rps8/rps8 lines Rps8/rps8 lines

PI 399073 PI 408211B Williams 1403 1404 1412 1413 37 50 58 143

Meiotic productsa

Dyadsb 27 0 0 18 9 12 0 0 0 0 0

Multinucleatedc 27 0 0 0 0 0 0 0 0 0 0

Triadsd 22 0 0 0 0 0 0 0 0 0 0

Multinucleated 22 0 0 0 0 0 0 0 0 0 0

Tetradse 137 149 81 61 86 95 69 10 84 77 111

Multinucleated 87 8 2 49 67 78 45 3 0 0 7

Pentadsf 16 0 0 0 0 0 0 0 0 0 0

Multinucleated 2 0 0 0 0 0 0 0 0 0 0

Pollen

Uninucleatedg grains 312 301 25 199 216 204 141 220 250 297 298

Binucleatedh grains 5 31 256 2 12 8 3 8 6 9 13

Sterile grains 8 4 4 5 0 8 6 7 3 1 4

Microcytesi 0 0 0 25 18 30 47 0 0 0 0

Type of meiotic products and the number of nuclei per cell were recorded for each genotype. The viability of the mature pollen was

determined by the presence of acetic carmine in the cytoplasma Set of microspores, classified according to the number of microspores in each setb Two microsporesc Number of sets in which at least one microspore contained more than one nucleusd Three microsporese Four microsporesf Five microsporesg Pre-mitotic microsporesh Microspore containing the generative celli Non-viable, small (\10 lm) meiotic products grains containing micronuclei

Euphytica (2011) 181:77–88 85

123

gave rise to the microcytes in the Williams 9 PI

399073 BC4F2:3 is not clear, although a similar

mechanism is suspected since micronuclei in the

tetrads were located close to the wall (Fig. 3j), and it

is unlikely that the cell wall in the small pollen grains

could have originated using the limited genetic

material inside them. The maintenance of micronu-

clei within the microspores could be the result of low

efficiency in the elimination process.

The identification of different types of meiotic

products in PI 399073 and RILs of the Williams 9 PI

399073 BC4F2:3 was striking. Although these abnor-

mal dyads, triads, tetrad, and pentads do not appear to

have an effect on pollen viability, this phenomenon

may not be uncommon as similar types of meiotic

products have been observed in other species includ-

ing a pentaploid accession of Brachiaria brizantha

(Risso-Pascotto et al. 2003). In the microsporogenesis

stage, micronuclei were formed and some remained

inside the microspores, while others were eliminated

as microcytes in a similar mechanism to the described

by Baptista-Giacomelli et al. (2000). The dyads and

triads formed in B. brizantha were produced by

failure in cytokinesis, these microspores developed

into 2 N pollen through reinstitution of nucleus. It is

possible that a similar process occurs in PI 399073 as

this type of pollen was observed in anthers of this

landrace. This type of meiotic behavior could limit

the breeding potential of a particular genotype if the

progeny exhibits these meiotic abnormalities. Preco-

cious pollen germination in soybean genotypes was

previously described by Kaur et al. (2005) as a

strategy that might facilitate a high degree of selfing

and interfere in hybridization efforts. This could

explain the production of pods from partially open

flowers observed in the cultivar Williams.

These findings are limited to the heterozygous

plants that were evaluated in this study, thus the

mechanism behind the meiotic abnormalities in PI

Fig. 3 Meiotic products in PI 399073: a multinucleate dyad,

b multinucleate tetrad, c multinucleated triad, d mononucleated

pentad

Fig. 4 Microspores

characteristics among

parental genotypes and one

RIL: a PI 408211B,

mononucleated grains; b PI

399073, sterile grain;

c Williams, binucleated

grain; d Williams, grain

germinating inside the

anther; e Line 1403

(BC4F2:3), microcyte grain

and f Line 37 (F4:5),

mononucleated grains

86 Euphytica (2011) 181:77–88

123

399073 and its progeny, and what role if any these

abnormalities play specifically in the high degree of

segregation distortion at the Rps8 locus still needs to

be explored. In this study, the meiotically abnormal

lines originated from a cross where PI 399073 was

used as pollen donor, whereas in the meiotically

normal lines PI 399073 was the pollen recipient; it is

unknown if abnormalities in megasporogenesis are

occurring, if the ovules were affected, this could

explain why the differences were found between

these two populations. Unfortunately lines from

reciprocal crosses were not available at the time of

this study. In the future, if these lines are available

they can be used to determine if the meiotic

abnormalities depend on the genotype used as donor

parent or on the geographic/genetic distance between

the parents. Future studies will focus on the Rps8

region and its association with the chromosome

fragments, laggards, micronuclei and microcytes.

This will only be possible if BAC clones from PI

399073 and Williams 82 located in this region are

identified and fully characterized.

Acknowledgments This project was supported by State and

Federal Funds appropriated to the Ohio Agricultural Research

and Development Center (OARDC), The Ohio State

University. Funding was also provided in part through

soybean check off dollars from Ohio Soybean Council, Iowa

Soybean Association, and United Soybean Board. We thank

Steven St. Martin and Ron Fioritto for making the crosses and

developing populations that generated the RILs evaluated on

this study, Sue Ann Berry for the phenotypic evaluation of the

RILs, and Dr. Tea Meulia, at the Molecular and Cellular

Imaging Center (MCIC) at OARDC, for assistance with

microscopy imaging. We also thank Dr. Randy Shoemaker,

Dr. Saghai Maroof and Dr. Steven St. Martin for critical

discussions throughout this course of study.

Open Access This article is distributed under the terms of the

Creative Commons Attribution Noncommercial License which

permits any noncommercial use, distribution, and reproduction

in any medium, provided the original author(s) and source are

credited.

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